Novel strigolactone analogues and their use

ABSTRACT

Novel compounds of formula (I) their use as germination trap for parasitic weeds, for the regulation of branching, tillering and root development, for enhancement of cambium growth, for the regulation of hyphal growth of mycorrhizal fungi and compositions comprising compounds of formula (I) and insecticides and/or fungicides.

The present invention relates to novel compounds and to the uses thereoffor combatting or controlling parasitic weeds and for the regulation ofcertain plant properties and mycorrhizal fungi-plant interaction.

In the recent past, a new class of carotenoid-derived compounds havebeen intensively studied and are commonly referred to as strigolactones.Strigolactones have been identified in the root exudates of plants andare known to stimulate seed germination of parasitic plants, such asStriga and Orobanche (which are amongst the main agricultural pestworldwide), and to induce hyphal growth of arbuscular mycorhizal (AM)fungi that form symbiotic structures (arbuscules) with the majority ofland plants. More recently strigolactones have been identified as planthormones regulating branching and cambium growth.

As shown in FIG. 1, the common feature of the strigolactones is thecombination of a tricyclic lactone comprising rings A, B and C connectedto a butenolide group (D) that also represents a lactone.

Rings A, B and D may carry a variety of substituents and may underliedifferent modifications, but all strigolactones share the common basicstructure shown in FIG. 1.

The infection with striga and orobanchaceae is responsible for annuallosses for farmers of billions of dollars all over the world. Around twothird of the area for cereal crop production in Africa are infected withstriga varieties. Alectra, a member of the orobanchaceae affectsdicotyledonous crops in large parts of Europe and North America and isresponsible for significant yield losses.

As shown in FIG. 1, the tricyclic lactone comprising rings A, B and Cand the butenolide D are connected through an enol ether bridge. Due tothis specific structure, all natural strigolactones are very susceptibleto nucleophilic attack (hydrolysis) and thus unstable in aqueoussystems. Furthermore, their solubility in aqueous systems is normallyvery low.

Furthermore, the compounds in accordance with FIG. 1 comprise an enonestructural element built by the double bond between rings C and D andthe keto group in ring C. This substructure also has a detrimentalinfluence on the stability and properties of the said compounds. Thisenone structure has hitherto been considered indispensible to achievethe desired activity.

In view of their great potential for the so-called suicide germinationof parasitic weeds (this means the germination in the absence of asuitable host plant which will ultimately lead to the death of theparasitic plant) thorough investigations have been undertaken todetermine the minimum structural requirements necessary to maintain thedesired germination stimulating activity.

Strigolactones have also been shown to play a role in establishingsymbiotic arbuscular mycorrhiza (AM), a symbiotic structure formed byplants and soilborne fungi. AM allows plants to obtain water andnutrients from the soil through the hyphae of the fungi and in turn theplants provide the fungi with photosynthates. AM is considered veryadvantageous for crop yield and represent the most widespread symbiosison earth. The establishment of AM requires extensive branching of thehyphae, which is induced by root-derived signals commonly referred to asbranching factor (BF). Strigolactones play a role in this process. Thestructural requirements for this desired activity are deemed to be verysimilar to those for the above mentioned suicide germination of theparasitic weeds.

Finally, it has been shown that strigolactones fulfil the criteria ofhormones, i.e. they require a receptor mediating their response, theyare active at very low concentrations and they can be transported in theplant over some distance. Various investigations have shown thatstrigolactones are involved in root and lateral root growth and inphoto-morphogenesis. Further, they control the growth of cambium,indicating an important role in wood production.

A number of structural studies have been carried out to get a betterunderstanding of the essential parts in the strigolactone molecule forachieving desired properties as referred to above. The replacement ofthe A-ring by an aromatic ring leads to the active analogue commonlyreferred to as GR24. Analogues lacking the A-ring (GR7) or both the A-and B-ring (GR5) still exhibit appreciable, albeit lower activities.

In contrast, omitting of the D-ring or the ABC enol ether results inbasically complete loss of the desired activity. An analogue consistingonly of a D-ring with an ethoxy group is also inactive. Based on thesestructure-activity data, it was concluded that an essential minimalrequirement is the presence of the structural elements of the D-ringconnected with an enone unit via the mentioned enol ether. As mentionedabove, this unit, however, causes an inherent low stability of theactive compounds in aqueous media combined with a low solubility. Thismakes their use in applications in the crop protection area difficult.

Zwanenburg et al (J. Agrif. Food Chem. 1992, 40, 1222-1229) describesstructural modifications of strigol analogues and analyzes the influenceof the B and C rings on the bioactivity. FIG. 2 on page 1225 shows amodified strigol analogue which again comprises an enone substructureinvolving the carbon atom of the double bond between the butenolidestructure and the remainder of the molecule, which is detrimental forthe stability of the compounds (as mentioned hereinbefore).

Brooks et al., ACS Symposium Series, 268, 437-444 discloses compounds 30a and 30 b which again have the undesired enone substructure.

Kondo et al., Biosci. Biotechnol. Biochem. 71, 11, 2781-2787 disclosesimino analogues of strigolactones which share inter alia a commonstructural element of an enone structure as outlined above.

Johnsson et al., J. Chem. Soc. Perkin Trans. I, No. 6, Jan. 1, 1981,pages 1734-1743 describes procedures for the preparation of synthetisanalogues of strigol, which share the common structural feature of anenone-substructure as described above.

In Sci. China Ser. C Life Sciences, 2009 52(8), 693-700 the authorsdescribe strigolactone derivatives with rings C and D as shown in FIG. 1and thus comprising the undesired enone-substructure as outlined above.

Accordingly, there was a need for strigolactone analogues or compoundshaving similar activity as the strigolactones described in the prior artwhile at the same time showing enhanced stability in aqueous media.

It was thus an object of the instant invention to provide novelcompounds showing beneficial properties similar to those ofstrigolactones, but having an improved stability, in particular againstnucleophilic attack/hydrolysis.

It was a further object of the instant invention to provide beneficialuses for such new compounds in the regulation of certain plantproperties.

These objects have been achieved with the novel compounds in accordancewith claim 1. Preferred compounds are described in detail hereinafterand are claimed in dependent claims.

The novel compounds in accordance with the instant invention arerepresented by the general formula I

wherein R^(a), R^(b) and R^(c), independently from each other,represent:

a hydrogen atom, a halogen atom, a nitro group, a cyano group, aformyloxy group, a formylamino group or a carbamate group,

a substituent-R¹, wherein R¹ represents C₁-C₈-alkyl-, C₂-C₈alkenyl,C₂-C₈-alkinyl, C₃-C₈-cycloalkyl or C₁-C₈-alkoxy, in each of which thehydrogen atoms may be partly replaced by other groups or atoms,

a substituent —OR², wherein R² represents a hydrogen atom, C₁-C₈-alkyl,C₂-C₈-alkenyl, C₂-C₈-alkinyl, C₁-C₈-alkylcarbonyl,C₁-C₈-alkylaminocarbonyl or C₁-C₈-alkoxycarbonyl, in each of which thehydrogen atoms may be partly replaced by other groups or atoms

a substituent —NR³R⁴, wherein R³ and R⁴, independently from each other,represent a hydrogen atom, C₁-C₈ alkyl, C₁-C₈-alkylcarbonyl,C₁-C₈-halogenoalkylcarbonyl, phenyl or benzyl, in each of which thehydrogen atoms may be partly replaced by other groups or atoms,

a substituent —(O)—R⁵, wherein R⁵ represents a hydrogen atom,C₁-C₈-alkyl or C₁-C₈-alkyloxy, in each of which the hydrogen atoms maybe partly replaced by other groups or atoms, —NH2, NHR⁵ or NR⁵R⁵ (wherethe two substituents R⁵ may be the same or different, —NR⁵(OH),

a substituent —S(O)_(n)—R⁶, wherein n is 0, 1 or 2 and R⁶ representsC₁-C₈-alkyl in which the hydrogen atoms may be partly replaced by othergroups or atoms, —NH₂, —NHR⁶ or NR⁶R⁶ (where the two substituents R⁶ maybe the same or different),

or a 4-, 5-, 6- or 7-membered heterocyclic ring comprising up to 4heteroatoms selected from nitrogen, oxygen or sulfur, where in each ofthese rings the hydrogen atoms may be partly replaced by other groups oratoms,

R^(d) represents

C₁-C₈ alkyl, C₂-C₈-alkenyl or C₂-C₈-alkinyl, wherein the hydrogen atomsmay be partly replaced by other groups or atoms,

R^(e) represents

a substituent R¹ as defined above, or —CH═CH—R⁷, wherein R⁷ represents ahydrogen atom, C₁-C₈-alkyl or a 4-, 5- 6- or 7-membered saturated orunsaturated, aromatic or non-aromatic carbocyclic or heterocyclic ringor a fused ring system containing more than one of those rings or

R^(d) and R^(e) together form a 4-, 5- 6- or 7-membered saturated orunsaturated, aromatic or non-aromatic carbocyclic or heterocyclic ringwhich may be fused to another saturated or unsaturated, aromatic ornon-aromatic carbocyclic or heterocyclic ring with the proviso that thecarbon atom carrying substituents R^(d) and R^(e) is not part of anenone-structural element, and

R^(f) represents

a hydrogen atom, a halogen atom, a nitro group, a cyano group orC₁-C₈-alkyl-, C₂-C₈ alkenyl, C₂-C₈-alkinyl or C₃-C₈-cycloalkyl.

The compounds of formula I, depending on the number of double bonds, mayexist in different isomers and formula I is intended to cover all suchisomers and to any mixtures thereof.

Due to the lack of the enone group such as represented by ring C, thestability of the novel compounds of formula I against nucleophilicattack/hydrolysis is improved compared to previously knownstrigolactones. Despite the lack of the enone substructure which hashitherto been considered indispensible for the desired activity of thestrigol analogues, the novel compounds in accordance with the presentinvention surpirsingly show the desired activity, which is contrary tothe expectations of the skilled person in view of the teaching of theprior art.

Preferred novel compounds in accordance with the present invention donot have the enol ether bridge between the butenolide D (lactone) and aring C—they do not contain a ring C at all but still retain the desiredactivity to a significant degree. Thus, they lack the structural elementwhich has hitherto been considered indispensible for the germinationstimulating activity and other desired properties. This was not to beexpected based on prior knowledge.

According to a first preferred embodiment, R^(a) and R^(b) are bothhydrogen and R^(c) is a substituent R¹, even more preferred R^(c) is aC₁ to C_(s)-alkyl group, in particular a C1-C4 alkyl group and mostpreferred a methyl group.

According to a further preferred embodiment, R^(e) represents asubstituent —CH═CH—R⁷, in which R⁷ represents a 4- to 7-memberedsaturated or unsaturated, aromatic or non aromatic carbocyclic orheterocyclic ring or a fused ring system containing more than one ofthose rings. More preferably R⁷ is a five- or six membered carbocyclicring which may be saturated or unsaturated, even more preferably R⁷ is acyclohexenyl ring. The ring R⁷ may be substituted by varioussubstituents and preferably by a hydroxyl group or C₁-C₈-alkyl,C₁-C₈-alkoxy or acetyl groups.

Still in another preferred embodiment, R^(e) represents a C₁-C₈ alkyl ora C₂-C₈-alkenylene group, optionally substituted with a hydroxyl groupor a carbonyl group or a cycloalkyl group which itself might besubstituted by e.g. one or more alkyl groups.

A particularly preferred group of compounds is represented by thefollowing formulae, wherein the cyclohexenyl or the phenyl ring mayoptionally be substituted by further methyl or hydroxyl groups.

Most particularly preferred is the following compound

The novel compounds of formula I can in principle be obtained by acoupling of the butenolide (lactone D) structure with the remaining partof the molecule via an enol ether bridge.

The synthesis of butenolides has been described in the literatureextensively so that further details are not necessary here. By way ofexample reference is made to Tetrahedron 2005, 61, 9896 (S. Ma and F.Yu, relating to the synthesis of γ-methylene-α,β-unsaturated γ-lactonesby Pd catalyzed cyclization of 3,4-alkadienoic acids) and C. Fu, S. MA,Eur. J. Org. Chem. 2005, 3942ff, relating to the iodolactonisation ofethyl-2,3-allenoates with Iodine in aqueous MeCN.

The novel compounds of formula I may also be obtained by biochemicalsynthesis in vitro using heterologously expressed CCD7, CCD8 and DWARF27enzymes or in vivo by expressing the genes coding for CCD7, CCD8 andDWARF 27 enzymes in a production host. Respective methods are known assuch to the skilled man and need not be further explained here.Microorganisms and moss may be mentioned as production hosts.

In the context of the studies leading to the present invention, it wasadditionally found that the combined activities of the carotinoidcleavage dioxygenases CCD7 and CCD8 lead to an unexpected product (seeExample 1).

The further study of the reaction mechanism revealed 9-cis-carotenoids,e.g. 9-cis-β-carotene, as precursors that are cleaved by CCD7 enzymes atthe 9′,10′ double bond leading to 9-cis-apo-10′-carotenals, e.g.9-cis-β-apo-10′-carotenal. Incubation of CCD8 enzymes with9-cis-β-apo-10′-carotenal or combined CCD7/CCD8 assays with9-cis-β-carotene lead to the compound with the structure IA

i.e. a particularly preferred compound in accordance with the instantinvention.

This product of combined CCD7/CCD8 activity and the identification of9-cis-β-carotene as substrate give rise to the assumption that thebiosynthetic pathway leading to 5-deoxystrigol (which is proposed to bethe precursor of all strigolactones) might be simpler than initiallysupposed.

In the course of the mechanistic studies, it was also found by theinventors that the protein DWARF-27, which is known to be involved inthe pathway to strigolactones in rice, is the isomerase required toproduce the precursor 9-cis-β-carotene from all-trans-β-carotene andthus a further aspect of the invention relates to the use of DWARF-27for isomerising all-trans-carotenes, e.g. for producing 9-cis-β-caroteneand other 9-cis-carotenes from their corresponding all-trans-isomers.

As already referred to above, the compounds of formula I and inparticular the compound obtained through the CCD7/CCD8 activity showproperties and behaviour like strigolactones but without the need ofhaving an enone structure coupled to a butenolide, which, as mentionedleads to an inherent instability against nucleophilic attack.

Different strategies, e.g. sanitation and hand weeding, crop rotation,improving soil fertility, soil treatment by fumigation and solarization,biological control, the use of selective herbicides, the use ofherbicide-resistant maize, chemicals inhibiting attachment (Strigaway®;BASF SE) and suicidal germination and breeding for resistance, have beenemployed to control root parasites. In general, these control strategiestarget either pre-attachment or post-attachment life-cycle stages ofparasitic plants. Since root parasites affect host developmentimmediately after attachment and cause important damage prior toparasite emergence, control strategies, which target pre-attachmentstages of the host-parasite interaction, are preferred. Being the firstcritical step in the interaction makes germination an important targetfor improved control measures. One advantage of this approach is thatthe inducing molecules are common to both Striga and Orobanche spp.Indeed, several control strategies based on the germination stimulantshave already been developed.

The compounds in accordance with the instant invention can be used assynthetic analogues to induce the germination of the seeds of theparasitic weeds. If these compounds are applied in the absence of asuitable host plant for the weed, the seeds will germinate and theparasitic weed will finally die due to the lack of a suitable host. Forexample, the compounds in accordance with the instant invention may beused in the seed treatment of seeds of non-hosts of the parasitic weeds,which may be used prior to planting the desired crop. Alternatively, onecould think of using compounds in accordance with the instant inventiontogether with a known herbicide for the parasitic weeds for thetreatment of seeds of the utility crops prior to transferring such seedsto the soil. If the crop itself is resistant against the herbicide, thecompounds in accordance with the instant invention would trigger thegermination of the parasitic weeds which would then be killed by theherbicide without negatively affecting the crop plant. All thesedifferent possibilities are included in the term germination trap andsame should be interpreted accordingly.

The compounds in accordance with the instant invention are also usefulto regulate, respectively to enhance, the hyphal growth of symbioticmycorrhizal fungi, which is a highly desirable symbiosis as sameincreases crop yields. This has in particular been observed inarbuscular mycorrhizal fungi like e.g. Gigaspora rosea.

A third beneficial usability of the compounds in accordance with theinstant invention is the regulation, respectively enhancement of cambiumgrowth, which is very important in wood production.

The compounds in accordance with the instant invention are also usefulfor the regulation of branching, tillering and root development ofplants.

The aerial plant architecture of aboveground shoots is determined by thepattern of shoot branching. The first step in shoot branching is thegeneration of axillary buds. Their development is mainly regulated bygenetics, but plants are able to modify their behaviour in response toenvironmental changes. The compounds in accordance with the instantinvention are useful to control the shoot branching of crops in adesired manner by applying these compounds to the crop in question.

Tiller is a stem produced at the base of grass plants and tilleringrefers to the production of such tillers, which process is stronglyinfluenced by the soil status. Tillering in rice (Oryza sativa L.) is animportant agronomic trait for grain production, and also a model systemfor the study of branching in monocotyledonous plants. Rice tiller is aspecialized grain-bearing branch that is formed on the unelongated basalinternode and grows independently of the mother stem (culm) by means ofits own adventitious roots. The compounds of the instant invention canbe used to regulate tillering in rice, thus increasing yields.

Furthermore, it has been found that compositions comprising at least onecompound of formula I and at least one insecticide and/or fungicideknown in the art show surprisingly increased efficiency for combating orcontrolling pests or fungi, in particular fungi detrimentally affectingplants. Processes for the treatment of fungi within or attached to thehuman body or within animals, to the extent same would constitute atherapeutic treatment of humans or animals, are excluded.

These compositions may provide a synergistic effect, which allows thereduction of the amount of chemicals spread and a reduction of thetreatment costs.

When used herein, the term synergistic effect means an efficacy of a newcombination of more than one compound exceeding the efficacy calculatedin accordance with the formula of Colby (Weeds 1967, 15, pages 20-22).This formula reads

E=x+y−(x*y)/100

in which E represents the expected efficiency (degree of inhibition ofdisease or pest) for the combination of two compounds at defined doses,x is the percentage of inhibition observed for the pest or disease bythe first compound of the combination (at a defined dose) and y is therespective efficiency for the second compound of the combination. Whenthe observed efficiency is higher than the expected efficiency accordingto this formula, this constitutes a synergistic effect.

The compositions containing at least one compound of formula I inaccordance with the instant invention and at least one insecticide orfungicide known in the art show such improvement compared to the singlecompounds in plant growth, vigor or yield of plants or crops orinsecticidal or fungicidal effect.

The weight ratio of the compounds in accordance with the instantinvention to the known insecticides or fungicides may cover a broadrange of from 0.5:1 to 1:10¹⁴, preferably of from 1:10² to 1:10⁸, andmore preferably of from 1:10³ to 1:10⁷, i.e. the content of the knowninsecticide or fungicide is usually much higher than the concentrationof the compounds in accordance with the instant invention.

Suitable insecticides are known to the skilled man and have beendescribed in the literature. They are generally classified in accordancewith their mode of action (to the extent same is known) asacetylcholione receptor agonists or antagonists, acetylcholinesteraseinhibitors, sodium channel modulators or sodium channel blockers,GABA-gated chloride channel antagonists, ecdysone agonists/disruptors,chloride channel activators, semicarbazones, juvenile hormone mimetics,acetylcholine receptor modulators, neirostoxin analogues, inhibitors oflipid biosynthesis, decouplers of oxidative phosphorylation, electrontransport inhibitors (site I, site II and site III), carboxamides,inhibitors of ATP-ase, inhibitors of oxidative phosphorylation,inhibitors of chitine biosynthesis, microbial disruptors of theintestinal membrane or ryanodin receptor antagonists. Furthermore, socalled biologicals, hormones or pheromones are known to the skilled manand there are also insecticides which are commercially used the mode ofaction however is not yet fully clarified.

The skilled man knows suitable insecticides of the aforementionedcategories suitable for the mixture compositions in accordance with theinstant invention. A good overview, including preferred insecticidalcompounds is given in WO 2008/152091, to which reference is made in thisregard.

Preferred insecticides useful in the compositions in accordance with thepresent invention are listed below:

organo(thio)phosphates: acephate, azamethiphos, azinphos-methyl,chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, diazinon,dichlorvos, dicrotophos, dimethoate, disulfoton, ethion, fenitrothion,fenthion, isoxathion, malathion, methamidophos, methidathion,methyl-parathion, mevinphos, monocrotophos, oxydemeton-methyl, paraoxon,parathion, phenthoate, phosalone, phosmet, phosphamidon, phorate,phoxim, pirimiphos-methyl, profenofos, prothiofos, sulprophos,tetrachlorvinphos, terbufos, triazophos, trichlorfon;

carbamates: alanycarb, aldicarb, bendiocarb, benfuracarb, carbaryl,carbofuran, carbosulfan, fenoxycarb, furathiocarb, methiocarb, methomyl,oxamyl, pirimicarb, propoxur, thiodicarb, triazamate;

pyrethroids: allethrin, bifenthrin, cyfluthrin, cyhalothrin,cyphenothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin,zeta-cypermethrin, deltamethrin, esfenvalerate, etofenprox,fenpropathrin, fenvalerate, imiprothrin, lambda-cyhalothrin, permethrin,prallethrin, pyrethrin I and II, resmethrin, silafluofen,tau-fluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin,profluthrin, dimefluthrin;

insect growth regulators: a) chitin synthesis inhibitors: benzoylureas:chlorfluazuron, cyramazin, diflubenzuron, flucycloxuron, flufenoxuron,hexaflumuron, lufenuron, novaluron, teflubenzuron, triflumuron;buprofezin, diofenolan, hexythiazox, etoxazole, clofentazine; b)ecdysone antagonists: halofenozide, methoxyfenozide, tebufenozide,azadirachtin; c) juvenoids: pyriproxyfen, methoprene, fenoxycarb; d)lipid biosynthesis inhibitors: spirodiclofen, spiromesifen,spirotetramat;

nicotinic receptor agonists/antagonists compounds: clothianidin,dinotefuran, flupyradifurone, imidacloprid, thiamethoxam, nitenpyram,acetamiprid, thiacloprid,1-2-chloro-thiazol-5-ylmethyl)-2-nitrimino-3,5-dimethyl-[1,3,5]triazinane;

GABA antagonist compounds: endosulfan, ethiprole, fipronil, vaniliprole,pyrafluprole, pyriprole,5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfinamoyl-1H-pyrazole-3-carbothioicacid amide;

macrocyclic lactone insecticides: abamectin, emamectin, milbemectin,lepimectin, spinosad, spinetoram;

mitochondrial electron transport inhibitor (METI) I acaricides:fenazaquin, pyridaben, tebufenpyrad, tolfenpyrad, flufenerim;

METI II and III compounds: acequinocyl, fluacyprim, hydramethylnon;

Uncouplers: chlorfenapyr;

oxidative phosphorylation inhibitors: cyhexatin, diafenthiuron,fenbutatin oxide, propargite;

moulting disruptor compounds: cryomazine;

mixed function oxidase inhibitors: piperonyl butoxide;

sodium channel blockers: indoxacarb, metaflumizone;

others: benclothiaz, bifenazate, cartap, flonicamid, pyridalyl,pymetrozine, sulfur, thiocyclam, flubendiamide, chlorantraniliprole,cyazypyr (HGW86), cyenopyrafen, flupyrazofos, cyflumetofen, amidoflumet,imicyafos, bistrifluoron, and pyrifluquinazon.

In some cases the following insecticides have proven to be particularlyuseful in combination with the compounds in accordance with the instantinvention: abamectin, aldicarb, beta-cyfluthrin, chlorpyrifos-E,clothianidin, cyromazine, deltamethrin, diflubenzuron, emamectin-b,fipronil, gamma-cyhalothrin, imidacloprid, L-cyhalothrin, methiocarb,pymetrozine, rynaxapyr, spinosad, spirodiclofen, spiromesifen,spirotetramate, tebufenozide, tebufenpyrad, tefluthrin, thiamethoxam andthiodicarb. These names are the so called INN designations of thecompounds well known to the skilled man in the art.

The compositions comprise at least one compound of formula I inaccordance with the instant invention and at least on insecticidecompound. However, it is also possible to use more than one of each ofthese groups of compounds to achieve even better results in certaincases.

Furthermore, the compositions may contain a fungicide in addition to aninsecticide; suitable fungicides are those also useful in mixtures ofcompounds of formula I with fungicides, which constitutes anotherembodiment of the instant invention described hereinafter.

Fungicides are known to the skilled man and described in the prior art.According to their mode of action, these compounds are categorized asinhibitors of nucleic acid synthesis, uncouplers, inhibitors of AA andprotein biosynthesis, inhibitors of mitosis and cell division,respiration inhibitors, inhibitors of ergosterol biosynthesis,inhibitors of melanine biosynthesis, host defence inducers, inhibitorsof lipid and membrane synthesis, inhibitors of cell wall synthesis,signal transduction inhibitors, ATP production inhibitors and otherknown fungicides which have a multisite action mechanism or the mode ofaction of which has not yet been fully elucidated.

Suitable as well as preferred fungicides for combinations with compoundsin accordance with the instant invention are disclosed in WO2008/152092, to which reference is made in this regard.

Furthermore, the following fungicides may be mentioned as examples forcombinations with the compounds in accordance with the present invention(as well for mixtures of the compounds solely with fungicides as well asfor mixtures comprisng insecticides and fungicides and a compound inaccordance with the present invention.

A) Respiration Inhibitors

Inhibitors of complex III at Q_(o), site (e.g. strobilurins):azoxystrobin, coumethoxystrobin, coumoxystrobin, dimoxystrobin,enestroburin, fenaminstrobin, fenoxystrobin/flufenoxystrobin,fluoxastrobin, kresoxim-methyl, metominostrobin, orysastrobin,picoxystrobin, pyraclostrobin, pyrametostrobin, pyraoxystrobin,trifloxystrobin,2-[2-(2,5-dimethyl-phenoxymethyl)-phenyl]-3-methoxy-acrylic acid methylester and2-(2-(3-(2,6-dichlorophenyl)-1-methyl-allylideneaminooxymethyl)-phenyl)-2-methoxyimino-N-methyl-acetamide,pyribencarb, triclopyricarb/chlorodin-carb, famoxadone, fenamidone;

inhibitors of complex III at Q_(i) site: cyazofamid, amisulbrom,[(3S,6S,7R,8R)-8-benzyl-3-[(3-acetoxy-4-methoxy-pyridine-2-carbonyl)amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl]2-methylpropanoate,[(3S,6S,7R,8R)-8-benzyl-3-[[3-(acetoxymethoxy)-4-methoxy-pyridine-2-carbonyl]amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl]2-methylpropanoate,[(3S,6S,7R,8R)-8-benzyl-3-[(3-isobutoxycarbonyloxy-4-methoxy-pyridine-2-carbonyl)amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl]2-methylpropanoate,[(3S,6S,7R,8R)-8-benzyl-3-[[3-(1,3-benzodioxol-5-ylmethoxy)-4-methoxy-pyridine-2-carbonyl]amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl]2-methylpropanoate;(3S,6S,7R,8R)-3-[[(3-hydroxy-4-methoxy-2-pyridinyl)carbonyl]amino]-6-methyl-4,9-dioxo-8-(phenylmethyl)-1,5-dioxonan-7-yl2-methylpropanoate

inhibitors of complex II (e.g. carboxamides): benodanil, bixafen,boscalid, carboxin, fenfuram, fluopyram, flutolanil, fluxapyroxad,furametpyr, isopyrazam, mepronil, oxycarboxin, penflufen, penthiopyrad,sedaxane, tecloftalam, thifluzamide,N-(4′-trifluoromethylthiobiphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(2-(1,3,3-trimethyl-butyl)-phenyl)-1,3-dimethyl-5-fluoro-1H-pyrazole-4-carboxamide,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide,3-(difluoromethyl)-1-methyl-N-(1,1,3-trimethylindan-4-yl)pyrazole-4-carboxamide,3-(trifluoromethyl)-1-methyl-N-(1,1,3-trimethylindan-4-yl)pyrazole-4-carboxamide,1,3-dimethyl-N-(1,1,3-trimethylindan-4-yl)pyrazole-4-carboxamide,3-(trifluoromethyl)-1,5-dimethyl-N-(1,1,3-trimethylindan-4-yl)pyrazole-4-carboxamide,3-(difluoromethyl)-1,5-dimethyl-N-(1,1,3-trimethylindan-4-yl)pyrazole-4-carboxamide,1,3,5-tri-methyl-N-(1,1,3-trimethylindan-4-yl)pyrazole-4-carboxamide;

other respiration inhibitors (e.g. complex I, uncouplers): diflumetorim,(5,8-difluoroquinazolin-4-yl)-{2-[2-fluoro-4-(4-trifluoromethylpyridin-2-yloxy)-phenyl]-ethyl}-amine;nitrophenyl derivates: binapacryl, dinobuton, dinocap, fluazinam;ferimzone; organometal compounds: fentin salts, such as fentin-acetate,fentin chloride or fentin hydroxide; ametoctradin; and silthiofam;

B) Sterol Biosynthesis Inhibitors (SBI Fungicides)

C14 demethylase inhibitors (DMI fungicides): triazoles: azaconazole,bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole,diniconazole-M, epoxiconazole, fenbuconazole, fluquinconazole,flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole,metconazole, myclobutanil, oxpoconazole, paclobutrazole, penconazole,propiconazole, prothioconazole, simeconazole, tebuconazole,tetraconazole, triadimefon, triadimenol, triticonazole, uniconazole,1-[rel-(2S;3R)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)-oxiranylmethyl]-5-thiocyanato-1H-[1,2,4]triazole,2-[rel-(2S,3R)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)-oxiranylmethyl]-2H-[1,2,4]triazole-3-thiol;imidazoles: imazalil, pefurazoate, prochloraz, triflumizol; pyrimidines,pyridines and piperazines: fenarimol, nuarimol, pyrifenox, triforine;

Delta14-reductase inhibitors: aldimorph, dodemorph, dodemorph-acetate,fenpropimorph, tridemorph, fenpropidin, piperalin, spiroxamine;

Inhibitors of 3-keto reductase: fenhexamid;

C) Nucleic Acid Synthesis Inhibitors

phenylamides or acyl amino acid fungicides: benalaxyl, benalaxyl-M,kiralaxyl, metalaxyl, metalaxyl-M (mefenoxam), ofurace, oxadixyl;

others: hymexazole, octhilinone, oxolinic acid, bupirimate,5-fluorocytosine, 5-fluoro-2-(p-tolylmethoxy)pyrimidin-4-amine,5-fluoro-2-(4-fluorophenylmethoxy)pyrimidin-4-amine;

D) Inhibitors of Cell Division and Cytoskeleton

tubulin inhibitors, such as benzimidazoles, thiophanates: benomyl,carbendazim, fuberidazole, thiabendazole, thiophanate-methyl;triazolopyrimidines:5-chloro-7-(4-methylpiperidin-1-yl)-6-(2,4,6-trifluorophenyl-[1,2,4]triazolo[1,5-a]pyrimidine

other cell division inhibitors: diethofencarb, ethaboxam, pencycuron,fluopicolide, zoxamide, metrafenone, pyriofenone;

E) Inhibitors of Amino Acid and Protein Synthesis

methionine synthesis inhibitors (anilino-pyrimidines): cyprodinil,mepanipyrim, pyrimethanil;

protein synthesis inhibitors: blasticidin-S, kasugamycin, kasugamycinhydrochloride-hydrate, mildiomycin, streptomycin, oxytetracyclin,polyoxine, validamycin A;

F) Signal Transduction Inhibitors

MAP/histidine kinase inhibitors: fluoroimid, iprodione, procymidone,vinclozolin, fenpiclonil, fludioxonil;

G protein inhibitors: quinoxyfen;

G) Lipid and Membrane Synthesis Inhibitors

Phospholipid biosynthesis inhibitors: edifenphos, iprobenfos,pyrazophos, isoprothiolane;

lipid peroxidation: dicloran, quintozene, tecnazene, tolclofos-methyl,biphenyl, chloroneb, etridiazole;

phospholipid biosynthesis and cell wall deposition: dimethomorph,flumorph, mandipropamid, pyrimorph, benthiavalicarb, iprovalicarb,valifenalate andN-(1-(1-(4-cyano-phenyl)ethanesulfonyl)-but-2-yl)carbamicacid-(4-fluorophenyl) ester;

compounds affecting cell membrane permeability and fatty acides:propamocarb, propamocarb-hydrochlorid

fatty acid amide hydrolase inhibitors:1-[4-[4-[5-(2,6-difluorophenyl)-4,5-dihydro-3-isoxazolyl]-2-thiazolyl]-1-piperidinyl]-2-[5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]ethanone

H) Inhibitors with Multi Site Action

inorganic active substances: Bordeaux mixture, copper acetate, copperhydroxide, copper oxychloride, basic copper sulfate, sulfur;

thio- and dithiocarbamates: ferbam, mancozeb, maneb, metam, metiram,propineb, thiram, zineb, ziram;

organochlorine compounds (e.g. phthalimides, sulfamides,chloronitriles): anilazine, chlorothalonil, captafol, captan, folpet,dichiofluanid, dichlorophen, flusulfamide, hexachlorobenzene,pentachlorphenole and its salts, phthalide, tolylfluanid,N-(4-chloro-2-nitro-phenyl)-N-ethyl-4-methyl-benzenesulfonamide;

guanidines and others: guanidine, dodine, dodine free base, guazatine,guazatine-acetate, iminoctadine, iminoctadine-triacetate,iminoctadine-tris(albesilate), dithianon,2,6-dimethyl-1H,5H-[1,4]dithiino[2,3-c:5,6-c′]dipyrrole-1,3,5,7(2H,6H)-tetraone;

I) Cell Wall Synthesis Inhibitors

inhibitors of glucan synthesis: validamycin, polyoxin B; melaninsynthesis inhibitors: pyroquilon, tricyclazole, carpropamid, dicyclomet,fenoxanil;

J) Plant Defence Inducers

acibenzolar-5-methyl, probenazole, isotianil, tiadinil, phosphonates:fosetyl, fosetyl-aluminum, phosphorous acid and its salts;

K) Unknown Mode of Action

bronopol, chinomethionat, cyflufenamid, cymoxanil, dazomet, debacarb,diclomezine, difenzoquat, difenzoquat-methylsulfate, diphenylamin,fenpyrazamine, flumetover, flusulfamide, flutianil, methasulfocarb,nitrapyrin, nitrothal-isopropyl, oxin-copper, proquinazid, tebufloquin,tecloftalam, triazoxide, 2-butoxy-6-iodo-3-propylchromen-4-one,N-(cyclo-propylmethoxyimino-(6-difluoro-methoxy-2,3-difluoro-phenyl)-methyl)-2-phenylacetamide,N′-(4-(4-chloro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl-N-methylformamidine,N′-(4-(4-fluoro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl-N-methylformamidine,N′-(2-methyl-5-trifluoromethyl-4-(3-trimethylsilanyl-propoxy)-phenyl)-N-ethyl-N-methylformamidine,N′-(5-difluoromethyl-2-methyl-4-(3-trimethylsilanyl-propoxy)-phenyl)-N-ethyl-N-methylformamidine,2-{1-[2-(5-methyl-3-trifluoromethyl-pyrazole-1-yl)-acetyl]-piperidin-4-yl}-thiazole-4-carboxylicacid methyl-(1,2,3,4-tetrahydro-naphthalen-1-yl)-amide,2-{1-[2-(5-methyl-3-trifluoromethyl-pyrazole-1-yl)-acetyl]piperidin-4-yl}-thiazole-4-carboxylicacid methyl-(R)-1,2,3,4-tetrahydro-naphthalen-1-yl-amide,1-[4-[4-[5-(2,6-difluorophenyl)-4,5-dihydro-3-isoxazolyl]-2-thiazolyl]-1-piperidinyl]-2-[5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]ethanone,methoxy-acetic acid 6-tert-butyl-8-fluoro-2,3-dimethyl-quinolin-4-ylester,N-Methyl-2-{1-[(5-methyl-3-trifluoromethyl-1H-pyrazol-1-yl)-acetyl]piperidin-4-yl}-N-[(1R)-1,2,3,4-tetrahydronaphthalen-1-yl]-4-thiazolecarboxamide,3-[5-(4-methylphenyl)-2,3-dimethyl-isoxazolidin-3-yl]-pyridine,3-[5-(4-chloro-phenyl)-2,3-dimethyl-isoxazolidin-3-yl]-pyridine(pyrisoxazole), N-(6-methoxy-pyridin-3-yl)cyclopropanecarboxylic acidamide,5-chloro-1-(4,6-dimethoxy-pyrimidin-2-yl)-2-methyl-1H-benzoimidazole,2-(4-chloro-phenyl)-N-[4-(3,4-dimethoxy-phenyl)-isoxazol-5-yl]-2-prop-2-ynyloxy-acetamide;

L) Antifungal biocontrol agents, plant bioactivators: Ampelomycesquisqualis (e.g. AQ 10® from Intrachem Bio GmbH & Co. KG, Germany),Aspergillus flavus (e.g. AFLAGUARD® from Syngenta, CH), Aureobasidiumpullulans (e.g. BOTECTOR® from bio-ferm GmbH, Germany), Bacillus pumilus(e.g. NRRL Accession No. B-30087 in SONATA® and BALLAD® Plus fromAgraQuest Inc., USA), Bacillus subtilis (e.g. isolate NRRL-Nr. B-21661in RHAPSODY®, SERENADE® MAX and SERENADE® ASO from AgraQuest Inc., USA),Bacillus subtilis var. amyloliquefaciens FZB24 (e.g. TAEGRO® fromNovozyme Biologicals, Inc., USA), Candida oleophila I-82 (e.g. ASPIRE®from Ecogen Inc., USA), Candida saitoana (e.g. BIOCURE® (in mixture withlysozyme) and BIOCOAT® from Micro Flo Company, USA (BASF SE) andArysta), Chitosan (e.g. ARMOUR-ZEN from BotriZen Ltd., NZ), Clonostachysrosea f. catenulata, also named Gliocladium catenulatum (e.g. isolateJ1446: PRESTOP® from Verdera, Finland), Coniothyrium minitans (e.g.CONTANS® from Prophyta, Germany), Cryphonectria parasitica (e.g.Endothia parasitica from CNICM, France), Cryptococcus albidus (e.g.YIELD PLUS® from Anchor Bio-Technologies, South Africa), Fusariumoxysporum (e.g. BIOFOX® from S.I.A.P.A., Italy, FUSACLEAN® from NaturalPlant Protection, France), Metschnikowia fruckola (e.g. SHEMER® fromAgrogreen, Israel), Microdochium dimerum (e.g. ANTIBOT® from Agrauxine,France), Phlebiopsis gigantea (e.g. ROTSOP® from Verdera, Finland),Pseudozyma flocculosa (e.g. SPORODEX® from Plant Products Co. Ltd.,Canada), Pythium oligandrum DV74 (e.g. POLYVERSUM® from Remeslo SSRO,Biopreparaty, Czech Rep.), Reynoutria sachlinensis (e.g. REGALIA® fromMarrone Biolnnovations, USA), Talaromyces flavus V117b (e.g. PROTUS®from Prophyta, Germany), Trichoderma asperellum SKT-1 (e.g. ECO-HOPE®from Kumiai Chemical Industry Co., Ltd., Japan), T. atroviride LC52(e.g. SENTINEL® from Agrimm Technologies Ltd, NZ), T. harzianum T-22(e.g. PLANTSHIELD® der Firma BioWorks Inc., USA), T. harzianum TH 35(e.g. ROOT PRO® from Mycontrol Ltd., Israel), T. harzianum T-39 (e.g.TRICHODEX® and TRICHODERMA 2000® from Mycontrol Ltd., Israel andMakhteshim Ltd., Israel), T. harzianum and T wade (e.g. TRICHOPEL fromAgrimm Technologies Ltd, NZ), T. harzianum ICC012 and T. viride ICC080(e.g. REMEDIER® WP from Isagro Ricerca, Italy), T. polysporum and T.harzianum (e.g. BINAB® from BINAB Bio-Innovation AB, Sweden), T.stromaticum (e.g. TRICOVAB® from C.E.P.L.A.C., Brazil), T. virens GL-21(e.g. SOILGARD® from Certis LLC, USA), T. viride (e.g. TRIECO® fromEcosense Labs. (India) Pvt. Ltd., Indien, BIO-CURE® F from T. Stanes &Co. Ltd., Indien), T. viride TV1 (e.g. T. viride TV1 from Agribiotecsrl, Italy), Ulocladium oudemansii HRU3 (e.g. BOTRY-ZEN® from Botry-ZenLtd, NZ)

The following fungicides have proved advantageous in certainapplications:

N-(2-(1,3-dimethylbutyl)phenyl)-5-fluoro-1,3-dimethyl-1H-pyrazole-4-carboxamide,metalaxyl, carbendazim, pencycuron, fenamidone, fluoxastrobin,pyrimethanil, iprodione, bitertanolo, fluquinconazole, ipconazole,prochloraz, epoxyconazole, prothioconazole, tebuconazole, triadimenol,triticonazole, carpropamid, tolylfluanid, fluopicolide, isotianil,N-(2-(1,1′-bi(cyclopropyl)-2-yl)phenyl-3-difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide,propamocarb, fosetylate,N-(2-(3-chloro)-5-trifluormethyl)-pyridin-2-yl)ethyl)-2-(trifluoromethyl)benzamide,fludioxonil, mefenoxam, pyraclostrobin, boscalid and azoxystrobin andmixtures thereof.

The compositions may contain one of more fungicides and one or morecompounds of formula I in accordance with the instant invention.

It has furthermore been found that the compounds in accordance with theinstant invention may also combined with known herbicides to providecompositions having increased efficacy in combating weeds compared toeither the activity of the known herbicide or the efficacy of thecompound of formula I. Suitable herbicides are known to the skilledperson and described in the literature.

The compositions in accordance with the instant invention may furthercomprise additional components such as carriers, supports, fillers,surfactants, protective colloids, adhesives, thickeners, stabilisers,thixotropic agents, penetration agents or sequestering agents. Ingeneral any additive may be contained which complies with the usualformulation techniques applied by the skilled persons in the preparationof respective compositions. Examples for such additives are given in WO2008/152091 or WO 2008/152092 to which reference is made for furtherdetails.

Without being specifically limited, the compositions in accordance withthe instant invention usually contain between 0.5 and 99% by weight ofcompounds of formula I and other active ingredients together, preferablyof from 10 to 90% by weight.

The compositions in accordance with the instant inventions may be usedin a variety of forms known to the skilled person in the art offormulation of insecticidal or fungicidal compositions. Again, detailsof such forms are given in WO 2008/152091 or WO 2008/152092 to whichreference is made for further details.

Details of methods for curatively or preventively controlling insectsand/or fungi and/or increasing the yield, growth or vigor of a plantapplying a composition in accordance with the instant invention via seedtreatment, foliar application, stem application, drench/dripapplication, to the seed, the plant or the soil are also given in WO2008/152091 or WO 2008/152092.

The compositions in accordance with the instant invention are welltolerated by plants and are environmentally friendly. Thus, thecompositions are suitable for improving the quality of the harvestedmaterial and for controlling animal pests encountered in agriculture, inforests, in gardens and leisure facilities, in the protection of storedproducts and in the hygiene sector.

The following examples show preferred embodiments of the instantinvention without limiting the scope thereof in accordance with theclaims.

The analytical methods referred to in the Examples were used as setforth below:

NMR Spectroscopy

For NMR analyses, a sample containing about 350 μg of the isolatedPsCCD7/8 product were dissolved in 500 μL of CD2Cl2. The NMR experimentswere performed on a Bruker AVANCE II spectrometer operation at a 1Hresonance frequency of 400.13 MHz. The instrument was equipped with a 5mm dual probe (BBI) for inverse detection with a z-gradient coil andwith pulse angles of 6.5 μs (¹H) and 13 μs (¹³C) respectively. BrukerTOPSPIN software (version 2.1, patch level 2) was used to acquire andprocess the NMR data. The measurements were carried out at roomtemperature (298 K) with the exception of the DOSY experiment measuredat 303 K (temperature controlled).

For the 1D ¹H experiment, the parameters were as follows: standardone-pulse experiment (zg), 32 transients, a spectral width of 9 ppm, adata size of 32K points, a relaxation delay 5 s, an acquisition time of4.55 s were used. For 2D ¹H-¹H-COSY the gradient-enhanced COSYexperiment for magnitude mode of detection (cosygqf), 32 scans, arelaxation delay of 1 s, an acquisition time of 0.57 s, a spectral widthin both dimensions (f2, f1) of 9 ppm, 4096 and 512 acquired data pointsin f2 and f1 respectively were used. Data were processed by zero filling(to 1024) in f1 and by using unshifted squared sine window functions inboth dimensions prior to 2D Fourier transformation.

For 2D ¹H-¹³C-HSQC, the gradient-enhanced HSQC experiment with carbonmultiplicity editing and echo-antiecho acquisition mode(hsqcedetgpsisp2.2), 64 transients, a relaxation time of 1.5 s, anacquisition time of 0.28 s, spectral widths of 9 ppm and 170 ppm (in f2and f1), with 2048 and 512 acquired data points respectively, were used.Data were processed by zero-filling (to 1024) and linear prediction inf1, and by using shifted squared sine window functions in bothdimensions prior to Fourier transformation.

For 2D ¹H-¹³C-HMBC, the gradient-enhanced HMBC experiment with twofoldlow-pass J-filter to suppress one-bond correlations and echo-antiechoacquisition mode (hmbcetgp12nd), 80 transients, a relaxation time of 1s, an acquisition time of 0.28 s, spectral widths (in f2 and f1) of 9ppm and 200 ppm with 2048 and 512 acquired data points respectively,were used. Data were processed by zero-filling (to 1024) and linearprediction in f1 and by using shifted squared sine window functions inboth dimensions prior to Fourier transformation.

For 1D ¹H-{¹H-}-NOE, the standard 1D NOE experiment using the secondchannel for selective irradiation of selected target protons (zgf2pr)was applied. For averaging unwanted long-term effects eightfold cyclingthrough the frequency list, with the acquisition of 8×32 transients forthe individual subspectra was performed. A spectral width of 9 ppm, adata size of 32K points, a relaxation and preirradiation delay of 3 s,and an acquisition time of 4.55 s were used.

¹H and ¹³C chemical shift prediction was performed using the ACD(Advanced Chemistry Development, Inc.) software, release 11.00 version11.02 (2008).

Enzymatic Assays with Crude and Purified Protein

Substrates were purified using thin-layer silica gel plates (Merck,Darmstadt, Germany). Plates were developed in lightpetroleum/diethylether/acetone (40:10:10, v/v/v). Substrates werescraped off in dim daylight and eluted with acetone. β-carotene wasobtained from Roth (Karlsruhe, Germany). 9-cis-β-carotene,13-cis-β-carotene and 15-cis-β-carotene were obtained from carotenature(Lupsingen, Switzerland).

Standard in vitro assays were performed in a total volume of 200 μl. 50μl of substrates (160 μM) in ethanol were mixed with 50 μl of ethanolicTriton X-100 (0.8%, v/v; Sigma, Deisenhofen, Germany), dried using avacuum centrifuge and then resuspended in 50 μl water. The preparedsubstrates were then mixed with 100 μl of 2× incubation buffercontaining 2 mM TCEP, 0.4 mM FeSO4, 200 mM Hepes-NaOH pH 8 and 2 mg/mlcatalase (Sigma, Deisenhofen, Germany). Purified Protein was then addedto a final concentration of 400 ng/μl. Crude assays were performed using50 μl of the soluble fractions of overexpressing cells. The assays wereincubated for 2 h at 28° C. Extraction was done by adding two volumes ofeach acetone and light petroleum/diethylether (1:4, v/v). Aftercentrifugation, the epiphase was collected.

CCD7/CCD8 double assays were performed in a total volume of 200 μl. 50μl of substrates (320 μM) in EtOH were mixed with 50 μl of ethanolicTriton X-100 (0.8%, v/v; Sigma, Deisenhofen, Germany), dried using avacuum centrifuge and then resuspended in 100 μl of 2× incubation buffercontaining 2 mM TCEP, 0.4 mM FeSO₄, 200 mM Hepes-NaOH pH 7.8 and 2 mg/mlcatalase (Sigma, Deisenhofen, Germany). Purified Protein was then addedto a final concentration of 200 ng/μl. Crude assays were performed usingeach 40 μl of the soluble fractions of overexpressing cells. The assayswere incubated for 3 h at 28° C. Extraction was done by adding twovolumes of each acetone and light petroleum/diethylether (1:4, v/v).After centrifugation, the epiphase was collected.

EXAMPLE 1 Biochemical Synthesis of Compound IA

In vitro incubation of CCD8 enzymes with 9-cis-β-apo-10′-caroteneresulted in a novel compound exhibiting an absorption maximum at around268 nm (the starting material has an absorption maximum at appr. 447nm), which was further analyzed after purification through two differentHPLC separation systems. Combined assays with a combination of CCD7 andCCD8 enzymes and using 9-cis-β-carotene as substrate led to the samenovel compound.

The purification of the novel compound by preparative HPLC was carriedout as follows:

A Waters system equipped with a photodiode array detector (model 996)was used to purify the PsCCD7/PsCCD8 product. The purification step wasperformed using a YMC-Pack C30-reversed phase column (250×4.6 mm i.d., 5μm; YMC Europe, Schermbeck, Germany) with the solvent systems B:methanol/water/tert-butylmethyl ether (60:20:2, v/v/v) and A:methanol/tert-butylmethyl ether (50:50, v/v). The column was developedat a flow-rate of 1 ml/min with a gradient from 100% B to 80% B within15 min, then to 0% B within 0.5 min and finally to 0% B and a flow-rateof 2 m/min within 0.5 min, maintaining the final conditions for another14 min. The collected fractions were dried using a rotary evaporator andresolved in methanol. The second purification step was performed using aMN Nucleosil 100 C18-reversed phase column (250×4 mm i.d., 10 μm;Macherey-Nagel, Düren, Germany) with the solvent systems B:methanol/water (20:80, v/v) and A: methanol. The column was developedfor 8 min with solvent B at a flow-rate of 1 ml/min, followed by alinear gradient from 100% B to 100% A within 1 min, maintaining thefinal conditions for another 10 min. For structure determination of thenovel compound, various NMR techniques were applied.

350 μg of the purified novel compound were dissolved in CD2Cl₂ andsubjected to 1D ¹H, 1D ¹H(¹H) NOE and three different 2D NMR techniques(2D ¹H-¹H-COSY, 2D ¹H-¹³C heteronuclear single quantum coherence and 2D¹H-¹³C heteronuclear multiple bond spectrometry). The 1D NOE experimentwas used to detect spatial proximity among protons. The 2D ¹H-¹H-COSYexperiment was applied for unraveling the homo-nuclear coupling networkand for assigning the corresponding ¹H signals to the individualprotons. Taking advantage of the 2D ¹H-¹³C-heteronuclear single quantumcoherence (HSQC) experiment dedicated for detecting one-bond (¹J_(CH))spin-spin interactions and its ability to differentiate among differentcarbon types (CH₃, CH₂, CH), these ¹H signals were correlated with the¹³C signals of directly bound carbons. The 2D ¹H-¹³C-heteronuclearmultiple bond correlation (HMBC) experiment, which detects long-rangecouplings between protons and carbons connected through two or threebonds (^(2,3)J_(CH)), served to assign the signals of quaternary carbonsand to independently prove the assignments of the proton-bearingcarbons. The analysis of the results allowed the unequivocal assignmentof the structure of formula IA to the novel compound. These results wereconfirmed through high resolution mass spectrometry yielding thecomposition C₁₉H₂₆O₃ and through UV spectroscopy indicating a maximum ofthree conjugated double bonds.

EXAMPLE 2 Isomerization Function of DWARF27

OsDWARF27 (an enzyme in rice) was expressed in fusion with thioredoxinand in vitro assays were performed with all-trans-β-carotene, usingcrude lysate of transformed E. coli cells. HPLC analysis revealed theconversion of the substrate into its 9-cis-isomer confirmed bycomparison with an authentic standard. In addition, combined incubationof DWARF27, CCD7 and CCD8 with all-trans-β-carotene led to the novelcompound of formula IA as obtained in Example 1, while controlsperformed with DWARF27 or with CCD7 and CCD8 did not show the formationof this compound.

This result confirmed the enzymatic function of DWARF27, which washitherto unclear, namely as an isomerase for all-trans carotenes to therespective cis-carotenes, in particular to form 9-cis-β-carotene fromall-trans-β-carotene.

EXAMPLE 3 Biological Activity of the Compound of Formula IA (asCharacterized in Example 1

Striga Seed Germination Assay

Pre-conditioning of Striga hermonthica seeds was carried out in a firststep to see the effect of germination stimulants. For this purpose cleanStriga seeds were surface sterilized with 2% sodium hypochlorite insterile water containing 0.4% of Tween-20. About 50 to 100 sterilizedseeds were spread on 9-mm diameter glass fibre filter paper discs.Twelve of these discs were placed in a 9-cm diameter Petri-dish onsterilized Whatman filter paper, moistened with 3.0 ml sterilized water.After sealing with parafilm, the Petri-dishes were placed in darkness inan incubator at 30° C. After 10 days of incubation, the discs withpreconditioned seeds were supposed to be ready to test germinationstimulant. Surplus moisture was evaporated from each petri dish inlaminar flow cabinet. The dry discs were then placed in anotherPetri-dish (six per Petri-dish) containing a filter paper ring (outerdiameter 9 cm, inner diameter 8 cm) moistened with 0.9 ml water. Variousconcentration of the compound of formula IA were applied (50 μl perdisc) to triplicate discs. GR24, a synthetic strigolactone analogue, wasapplied as positive control. Subsequently, the Striga seeds wereincubated in darkness at 30° C. for another 48 hours and germination(seeds with radicle protruding through the seed coat) scored with thehelp of a binocular microscope.

The compound of formula IA as obtained in Example in concentrations offrom 0.03 to 330 μM resulted in germination of the seeds in the range offrom 2 to 62%, which is an activity in this regard of the same order ofmagnitude as the known synthetic strigolactone analogue GR 24, whichyielded a germination activity of appr. 50% at a concentration of 3.3 μMunder the same conditions.

This result shows that compound IA can be used for germination of Strigavarieties.

Rice Tillering assay

Rice seeds were surface sterilized with 2% sodium hypochlorite. Theseeds were allowed to germinate in a Petri dish on a moistened filterpaper. In the meanwhile bottom of 1.5 ml eppendorf tube was cut andfilled with 0.6% phyto-agar. One pre-germinated seed was planted in thecenter of the tube on solid phytoagar and this tube was fitted in thecap of 10 ml plastic tube. The 10 ml tube was filled with modifiedHoagland nutrient solution. After two weeks of planting, the compound offormula IA (2 μM) was added through nutrient solution to half of thetubes of each rice cultivars/mutants and the remaining half were kept ascontrol. The nutrient solution with or without the compound of formulaIA was supplied daily. Number of tillers per plant was counted at 24hours interval up to 12 days of carlactone application.

In addition to its activity in inducing seed germination of parasiticplants, the compound of formula IA also exerted the function ofstrigolactone in regulating tillering of rice. Treatment with thiscompound led to a reduction of tiller number of the ccd8 rice mutant(d10) comparable to that shown by the strigolactone analogue GR24.

The results of the foregoing examples show that the novel compounds inaccordance with the instant invention can replace strigolactones in anumber of interesting applications.

1. Compounds of general formula I

wherein R^(a), R^(b) and R^(c), independently from each other,represent: a hydrogen atom, a halogen atom, a nitro group, a cyanogroup, a formyloxy group, a formylamino group or a carbamate group, asubstituent R¹, wherein R¹ represents C₁-C₈-alkyl-, C₂-C₈ alkenyl,C₂-C₈-alkinyl, C₃-C₈-cycloalkyl or C₁-C₈-alkoxy, in each of which thehydrogen atoms may be partly replaced by other groups or atoms, asubstituent —OR², wherein R² represents a hydrogen atom, C₁-C₈-alkyl,C₂-C₈-alkenyl, C₂-C₈-alkinyl, C₁-C₈-alkylcarbonyl,C₁-C₈-alkylaminocarbonyl or C₁-C₈-alkoxycarbonyl, in each of which thehydrogen atoms may be partly replaced by other groups or atoms asubstituent —NR³R⁴, wherein R³ and R⁴, independently from each other,represent a hydrogen atom, C₁-C₈ alkyl, C₁-C₈-alkylcarbonyl,C₁-C₈-halogenoalkylcarbonyl, phenyl or benzyl, in each of which thehydrogen atoms may be partly replaced by other groups or atoms, asubstituent —C(O)—R⁵, wherein R⁵ represents a hydrogen atom, C₁-C₈-alkylor C₁-C₈-alkyloxy, in each of which the hydrogen atoms may be partlyreplaced by other groups or atoms, —NH2, NHR⁵ or NR⁵R⁵ (where the twosubstituents R⁵ may be the same or different, —NR⁵(OH), a substituent—S(O)n-R⁶, wherein n is 0, 1 or 2 and R6 represents C₁-C₈-alkyl in whichthe hydrogen atoms may be partly replaced by other groups or atoms,—NH₂, —NHR⁶ or NR⁶R⁶ (where the two substituents R⁶ may be the same ordifferent), or a 4-, 5-, 6- or 7-membered heterocyclic ring comprisingup to 4 heteroatoms selected from nitrogen, oxygen or sulfur, where ineach of these rings the hydrogen atoms may be partly replaced by othergroups or atoms, R^(d) represents C₁-C₈ alkyl, C₂-C₈-alkenyl orC₂-C₈-alkinyl, wherein the hydrogen atoms may be partly replaced byother groups or atoms, R^(e) represents a substituent R¹ as definedabove, or —CH═CH—R⁷, wherein R⁷ represents a hydrogen atom, C₁-C₈-alkylor a 4-, 5- 6- or 7-membered saturated or unsaturated, aromatic ornon-aromatic carbocyclic or heterocyclic ring or a fused ring systemcontaining more than one of those rings R^(d) and R^(e) together form a4-, 5- 6- or 7-membered saturated or unsaturated, aromatic ornon-aromatic carbocyclic or heterocyclic ring, which may be fused toanother saturated or unsaturated, aromatic or non-aromatic carbocyclicor heterocyclic ring with the proviso that the carbon atom carryingsubstituents R^(d) and R^(e) is not part of an enone-structural element,and R^(f) represents a hydrogen atom, a halogen atom, a nitro group, acyano group or C₁-C₈-alkyl-, C₂-C₈ alkenyl, C₂-C₈-alkinyl orC₃-C₈-cycloalkyl.
 2. Compounds in accordance with claim 1 wherein R^(a)and R^(b) are hydrogen and R^(c) is a substituent R¹.
 3. Compounds inaccordance with claim 1 wherein R^(c) is C₁-C₈-alkyl.
 4. Compounds inaccordance with claim 1, represented by the following formulae


5. A compound in accordance with claim 1 represented by formula IA


6. (canceled)
 7. A method of controlling the germination of parasiticroot plants wherein a compound in accordance with claim 1 is used. 8.(canceled)
 9. A process for regulating branching, tillering and rootdevelopment of plants wherein a compound in accordance with claim 1 isused.
 10. (canceled)
 11. A process for regulating hyphal growth ofsymbiotic mycorrhizal fungi wherein a compound in accordance with claim1 is used.
 12. A composition comprising at least one compound inaccordance with claim 1 and at least one insecticide compound.
 13. Acomposition comprising at least one compound in accordance with claim 1and at least one fungicide compound.
 14. (canceled)
 15. A process forcombatting or controlling pests wherein a composition in accordance withclaim 12 is used.
 16. (canceled)
 17. A process for combatting orcontrolling fungi, in particular fungi affecting plants, wherein acomposition in accordance with claim 13 is used.
 18. (canceled)
 19. Aprocess for the isomerisation of all-trans carotenes wherein DWARF-27 isused.
 20. A process for the biochemical synthesis of compounds offormula I in vitro using heterologously expressed CCD7, CCD8 and DWARF27enzymes.
 21. A process for the biochemical synthesis of compounds offormula I in vivo by expressing the genes coding for CCD7, CCD8 andDWARF27 enzymes in a production host.